Complex formation of apo-enzyme, coenzyme and substrate of d-amino acid oxidase : I. Kinetic analysis using indicators

Abstract

1. I. It was found that both riboflavin-5′-monosulfate (FMS) and adenosine-5′-monosulfate (AMS) compete with FAD for binding with apo- d-amino acid oxidase but not with each other, which suggests the binding of the FMN as well as the AMP moieties of the FAD molecule with the apo-protein. 2. 2. Using FMS and AMS as indicators, p-aminobenzoic acid (PABA) was found to be a competitor with the FMN part of FAD. It was considered likely that the phenylamino group affects the binding point of the FMN part of FAD with the apo-protein. The dissociation constant of the apo-protein-PABA complex calculated from the competition of PABA with FAD for the protein appeared to be 7.3·10 −3 M. In the presence of AMS, this dissociation constant decreased to 2.5·10 −3 M, i. e., the affinity of PABA to the protein increased, which indicates an interaction between the two binding sites. 3. 3. p-Chloromercuribenzoate (PCMB) was found to be a competitor of the AMP moiety of FAD. This action was attributed to the binding of the SH-reactive group of PCMB with the sulfhydryl group of the apo-protein, which suggests that the AMP moiety of FAD combined with sulfhydryl group of the apo-protein. The dissociation constant of the complex of PCMB and apo-protein was calculated to be 1.6·10 −7 M. In the presence of FMS, this constant decreased to 8.5·10 −8 M, also suggesting an interaction of the binding sites of the apo-protein.